mRNA FOR USE IN TREATMENT OF HUMAN GENETIC DISEASES

ABSTRACT

Compositions for modulating the expression of a protein in a target cell comprising at least one RNA molecule which comprises at least one modification 5 conferring stability to the RNA, as well as related methods, are disclosed.

RELATED APPLICATIONS

The subject application claims the benefit of U.S. ProvisionalApplication Ser. No. 61/418,389, filed Nov. 30, 2010, the entireteachings of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Novel approaches and therapies are still needed for the treatment ofprotein and enzyme deficiencies, particularly strategies and therapieswhich overcome the challenges and limitations associated with theadministration of nucleic acids and the transfection of target cells.Additional approaches which modulate or supplement the expression of adeficient protein or enzyme and thus ameliorate the underlyingdeficiency would be useful in the development of appropriate therapiesfor associated disorders.

SUMMARY OF THE INVENTION

Disclosed herein are compositions for modulating the expression of aprotein in a target cell, wherein said composition comprises at leastone RNA molecule and a transfer vehicle and wherein the RNA comprises atleast one modification which confers stability to the RNA. In someembodiments the RNA molecule is selected from the group consisting ofmRNA, miRNA, snRNA, and snoRNA. In some embodiments the RNA moleculecomprises more than one modification which confers stability to the RNAmolecule.

In some embodiments the RNA molecule comprises a modification of the 5′untranslated region of said RNA molecule; for example, the modificationcan comprise all or a partial sequence of a CMV immediate-early 1 (IE1)gene. In some embodiments the partial sequence of the CMVimmediate-early 1 (IE1) gene comprises SEQ ID NO: 2 or SEQ ID NO: 1. Inother embodiments the modification comprises the inclusion of a poly Atail or a Cap1 structure.

In some embodiments the RNA molecule comprises a modification of the 3′untranslated of said RNA molecule; for example, the modification cancomprise the inclusion of a sequence encoding human growth hormone (hGH)(e.g., SEQ ID NO: 3).

In some embodiments the RNA encodes ornithine carbamoyltransferase,alpha galactosidase, or erythropoietin.

Also disclosed are methods of treating a subject deficient in a protein,comprising administering a composition comprising an mRNA and a transfervehicle, wherein the mRNA encodes a functional protein corresponding tothe protein which is deficient in the subject, and wherein the mRNAcomprises at least one modification which confers stability to theadministered mRNA. Preferably following expression of said mRNA by atarget cell a functional protein is produced. In some embodiments thefunctional protein is secreted from said target cell. In someembodiments the mRNA encodes ornithine carbamoyltransferase, alphagalactosidase, or erythropoietin.

Also disclosed are methods of intracellular delivery of nucleic acidsthat are capable of correcting existing genetic defects and/or providingbeneficial functions to one or more target cells. Following successfuldelivery to target tissues and cells, the compositions and nucleic acidsof the present invention transfect that target cell and the nucleicacids (e.g., mRNA) can be translated into the gene product of interest(e.g., a functional protein or enzyme) or can otherwise modulate orregulate the presence or expression of the gene product of interest.

The compositions and methods provided herein are useful in themanagement and 30 treatment of a large number of diseases, in particulardiseases which result from protein and/or enzyme deficiencies.Individuals suffering from such diseases may have underlying geneticdefects that lead to the compromised expression of a protein or enzyme,including, for example, the non-synthesis of the protein, the reducedsynthesis of the protein, or synthesis of a protein lacking or havingdiminished biological activity. For example, the methods andcompositions provided herein are useful for the treatment of the ureacycle metabolic disorders that occur as a result of one or more defectsin the biosynthesis of enzymes involved in the urea cycle. The methodsand compositions provided herein are also useful in various in vitro andin vivo applications in which the delivery of a nucleic acid (e.g.,mRNA) to a target cell and transfection of that target cell are desired.

In one embodiment, the compositions provided herein may comprise anucleic acid, a transfer vehicle and an agent to facilitate contactwith, and subsequent transfection of a target cell. The nucleic acid canencode a clinically useful gene product or protein. For example, thenucleic acid may encode a functional urea cycle enzyme. In preferredembodiments, the nucleic acid is RNA, or more preferably mRNA encoding afunctional protein or enzyme.

In some embodiments, compositions and methods for increasing expressionof a functional protein or enzyme in a target cell are provided. Forexample, the compositions and methods provided herein may be used toincrease the expression of a urea cycle enzyme (e.g, OTC, CPS1, ASS1,ASL or ARG1). In some embodiments, the composition comprises an mRNA anda transfer vehicle. In some embodiments, the mRNA encodes a urea cycleenzyme. In some embodiments the mRNA can comprise one or moremodifications that confer stability to the mRNA (e.g., compared to awildtype or native version of the mRNA) and/or may also comprise one ormore modifications relative to the wild-type which correct a defectimplicated in the associated aberrant expression of the protein. Forexample, the nucleic acids of the present invention may comprisemodifications to one or both of the 5′ and 3′ untranslated regions. Suchmodifications may include, but are not limited to, the inclusion of allor a partial sequence of a promoter (e.g., a viral promoter sequencesuch as the cytomegalovirus (CMV) immediate-early 1 (IE1) gene), a polyA tail, a Cap I structure or a sequence encoding all or a portion ofhuman growth hormone (hGH)). Notably in this context the promotersequence does not function as a promoter, as it is joined to an RNAsequence, e.g., an mRNA sequence rather than a DNA sequence. Suchmodifications may further comprise the addition of nucleic acids whichattract ribosomes (e.g., by virtue of their nucleic acid composition orsecondary structure).

Methods of treating a subject, wherein the subject has a protein orenzyme deficiency, are also provided. The methods can compriseadministering a composition provided herein. For example, methods oftreating or preventing conditions in which production of a particularprotein and/or utilization of a particular protein is inadequate orcompromised are provided. In one embodiment, the methods provided hereincan be used to treat a subject having a deficiency in one or more ureacycle enzymes. The method can comprise contacting and transfectingtarget cells or tissues (such as hepatocytes that are deficient in oneor more urea cycle enzymes) with a composition provided herein, whereinthe nucleic acid encodes the deficient urea cycle enzyme. In thismanner, the expression of the deficient enzyme in the target cell isincreased, which in turn is expected to ameliorate the effects of theunderlying enzyme deficiency. The protein or enzyme expressed by thetarget cell from the translated mRNA may be retained within the cytosolof the target cell or alternatively may be secreted extracellularly. Insome embodiments, the nucleic acid is an mRNA. In some embodiments, themRNA comprises a modification that confers stability to the mRNA (e.g.,when compared to the wild-type or native version of the mRNA). Forexample, the mRNA encoding a functional enzyme may comprise one or moremodifications to one or both of the 5′ and 3′ untranslated regions.

Methods of expressing a functional protein or enzyme (e.g., a urea cycleenzyme) in a target cell are also provided. In some embodiments, thetarget cell is deficient in the protein or enzyme, e.g., a urea cycleenzyme. The methods comprise contacting the target cell with acomposition comprising an mRNA and a transfer vehicle. Followingexpression of the protein or enzyme encoded by the mRNA, the expressedprotein or enzyme may be retained within the cytosol of the target cellor alternatively may be secreted extracellularly. In some embodiments,the mRNA encodes a urea cycle enzyme. In some embodiments the mRNA cancomprise one or more modifications that confer stability to the mRNAand/or may also comprise one or more modifications relative to thewild-type that correct a defect implicated in the associated aberrantexpression of the protein. In some embodiments, the compositions andmethods of the present invention rely on the target cells to express thefunctional protein or enzyme encoded by the exogenously administerednucleic acid (e.g., mRNA). Because the protein or enzyme encoded by theexogenous mRNA are translated by the target cell, the proteins andenzymes expressed may be characterized as being less immunogenicrelative to their recombinantly prepared counterparts.

Also provided are compositions and methods useful for facilitating thetransfection and delivery of one or more nucleic acids (e.g., mRNA) totarget cells. For example, the compositions and methods of the presentinvention contemplate the use of targeting ligands capable of enhancingthe affinity of the composition to one or more target cells. In oneembodiment, the targeting ligand is apolipoprotein-B or apolipoprotein-Eand corresponding target cells express low-density lipoproteinreceptors, thereby facilitating recognition of the targeting ligand. Themethods and compositions of the present invention may be used topreferentially target a vast number of target cells. For example,contemplated target cells include, but are not limited to, hepatocytes,epithelial cells, hematopoietic cells, epithelial cells, endothelialcells, lung cells, bone cells, stem cells, mesenchymal cells, neuralcells, cardiac cells, adipocytes, vascular smooth muscle cells,cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells,synovial lining cells, ovarian cells, testicular cells, fibroblasts, Bcells, T cells, reticulocytes, leukocytes, gramtlocytes and tumor cells.

The above discussed and many other features and attendant advantages ofthe present invention will become better understood by reference to thefollowing detailed description of the invention when taken inconjunction with the accompanying examples. The various embodimentsdescribed herein are complimentary and can be combined or used togetherin a manner understood by the skilled person in view of the teachingscontained herein.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the nucleotide sequences of 5′ CMV sequences (SEQ ID NO: 2and SEQ ID NO: 1) and a 3′ hGLI sequence (SEQ ID NO: 3) which may, inparticular embodiments, be used to flank an mRNA sequence of interest.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are compositions that facilitate the delivery ofnucleic acids to, and the subsequent transfection of, target cells. Inparticular, the compositions provided herein are useful for thetreatment of diseases which result from the deficient production ofproteins and/or enzymes. For example, suitable diseases that may betreated are those in which a genetic mutation in a particular genecauses affected cells to not express, have reduced expression of, or toexpress a non-functional product of that gene. Contacting such targetcells with the compositions of the present invention such that thetarget cells are transfected with a nucleic acid encoding a functionalversion of the gene product allows the production of a functionalprotein or enzyme product this is useful in the treatment of suchdeficiency. In particular embodiments, the compositions comprise one ormore modifications that confer stability to the nucleic acid (e.g.,mRNA) and/or comprise one or more modifications relative to thewild-type that correct a defect implicated in the associated aberrantexpression of the protein.

Provided herein are compositions for modulating the expression of aprotein in a target cell. In some embodiments, the composition comprisesan RNA molecule and a transfer vehicle. In preferred embodiments thecomposition comprises an mRNA molecule comprising one or moremodifications that confer stability and/or increased half-life in vivoto the mRNA and/or comprising one or more modifications relative to thewild-type that correct a defect implicated in the associated aberrantexpression of the protein. In some embodiments, the mRNA of thecomposition can be modified to impart enhanced stability (e.g., relativeto the wild-type version of the mRNA and/or the version of the mRNAfound endogenously in the target cell). For example, the mRNA of thecomposition can include a modification compared to a wild-type versionof the mRNA, wherein the modification confers stability to the mRNA ofthe composition.

Provided herein are methods of and compositions for modulating the levelof mRNA and/or the expression of protein in a subject. In someembodiments, the compositions provided herein are capable of modulatingthe expression of a particular protein by increasing the level/amount ofmRNA encoding that protein in a target cell or tissue. Delivery of anmRNA encoding the desired protein can be achieved as described herein,and the mRNA is translated by the target cell to produce protein. Insome embodiments, the mRNA of the composition is more stable (e.g., haslimited or reduced nuclease susceptibility) compared to a wild-typeand/or endogenous version of the nucleic acid, as the mRNA comprises oneor more modifications that confer stability and/or increased half-lifein vivo to the mRNA and/or comprises one or more modifications relativeto the wild-type that correct a defect implicated in an associatedaberrant expression of the protein.

In other embodiments, the compositions provided herein are capable ofmodulating the expression of a particular protein by decreasingexpression of mRNA encoding that protein in a target cell or tissue. Forexample, in one embodiment, the composition comprises a miRNA or anucleic acid encoding miRNA where the miRNA is capable of reducing oreliminating expression of a particular mRNA in a target cell. In someembodiments, the nucleic acid of the composition is more stable (e.g.,has limited or reduced nuclease susceptibility) compared to a wild-typeand/or endogenous version of the nucleic acid.

As used herein, the term “nucleic acid” refers to genetic material(e.g., oligonucleotides or polynucleotides comprising DNA or RNA). Insome embodiments, the nucleic acid of the compositions is RNA. SuitableRNA includes mRNA, siRNA, miRNA, snRNA and snoRNA. Contemplated nucleicacids also include large intergenic non-coding RNA (lincRNA), whichgenerally do not encode proteins, but rather function, for example, inimmune signaling, stem cell biology and the development of disease.(See, e.g., Guttman, et al., 458: 223-227 (2009); and Ng, et al., NatureGenetics 42: 1035-1036 (2010), the contents of which are incorporatedherein by reference). In a preferred embodiment, the nucleic acids ofthe invention include RNA or stabilized RNA encoding a protein orenzyme. The present invention contemplates the use of such nucleic acids(and, in particular, RNA or stabilized RNA) as a therapeutic capable offacilitating the expression of a functional enzyme or protein, andpreferably the expression of a functional enzyme of protein in which asubject is deficient (e.g., a urea cycle enzyme). The term “functional”,as used herein to qualify a protein or enzyme, means that the protein orenzyme has biological activity, or alternatively is able to perform thesame or a similar function as the native or normally-functioning proteinor enzyme. The subject nucleic acid compositions of the presentinvention are useful for the treatment of various metabolic or geneticdisorders, and in particular those genetic or metabolic disorders whichinvolve the non-expression, misexpression or deficiency of a protein orenzyme.

In the context of the present invention the term “expression” is used ina broad sense to refer to either the transcription of a specific gene ornucleic acid into at least one mRNA transcript, or the translation of atleast one mRNA or nucleic acid into a protein or enzyme. For example,contemplated by the present invention are compositions which compriseone or more mRNA nucleic acids which encode functional proteins orenzymes, and in the context of such mRNA nucleic acids, the tennexpression refers to the translation of such mRNA to produce the proteinor enzyme encoded thereby.

The nucleic acids provided herein can be introduced into cells ortissues of interest. In some embodiments, the nucleic acid is capable ofbeing translated (e.g., the translation of the encoded protein or enzymefrom a synthetic or exogenous mRNA transcript) or otherwise capable ofconferring a beneficial property to the target cells or tissues (e.g.,reducing the expression of a target nucleic acid or gene). The nucleicacid may encode, for example, a hormone, enzyme, receptor, polypeptide,peptide or other protein of interest. A nucleic acid may also encode asmall interfering RNA (siRNA) or antisense RNA for the purpose ofdecreasing or eliminating expression of an endogenous nucleic acid orgene. In one embodiment of the present invention, the nucleic acid(e.g., mRNA encoding a deficient protein or enzyme) may optionally havechemical or biological modifications which, for example, improve thestability and/or half-life of such nucleic acid or which improve orotherwise facilitate translation.

The nucleic acids of the present invention may be natural or recombinantin nature and may exert their therapeutic activity using either sense orantisense mechanisms of action.

Also contemplated by the present invention is the co-delivery of one ormore unique nucleic acids to target cells, for example, by combining twounique nucleic acids into a single transfer vehicle. In one embodimentof the present invention, a therapeutic first nucleic acid, such as mRNAencoding galactose-1-phosphate uridyltransferase (GALT), and atherapeutic second nucleic acid, such as mRNA encoding galatokinase(GALK), may be formulated in a single transfer vehicle and administered(e.g., for the treatment of galactosemia). The present invention alsocontemplates co-delivery and/or co-administration of a therapeutic firstnucleic acid and a second nucleic acid to facilitate and/or enhance thefunction or delivery of the therapeutic first nucleic acid. For example,such a second nucleic acid (e.g., exogenous or synthetic mRNA) mayencode a membrane transporter protein that upon expression (e.g.,translation of the exogenous or synthetic mRNA) facilitates the deliveryor enhances the biological activity of the first nucleic acid.Alternatively, the therapeutic first nucleic acid may be administeredwith a second nucleic acid that functions as a “chaperone” for example,to direct the folding of either the therapeutic first nucleic acid orendogenous nucleic acids.

Also contemplated is the delivery of one or more therapeutic nucleicacids to treat a single disorder or deficiency, wherein each suchtherapeutic nucleic acid functions by a different mechanism of action.For example, the compositions of the present invention may comprise atherapeutic first nucleic acid which, for example, is administered tocorrect an endogenous protein or enzyme deficiency, and which isaccompanied by a second nucleic acid, which is administered todeactivate or “knock-down” a malfunctioning endogenous nucleic acid andits protein or enzyme product. Such nucleic acids may encode, forexample mRNA and siRNA.

The nucleic acids provided herein, and in particular the mRNA nucleicacids provided herein, preferably retain at least some ability to betranslated, thereby producing a functional protein or enzyme within atarget cell. Accordingly, the present invention relates to theadministration of a stabilized nucleic acid (e.g., mRNA which has beenstabilized against in vivo nuclease digestion or degradation) tomodulate the expression of a gene or the translation of a functionalenzyme or protein within a target cell. In a preferred embodiment of thepresent invention, the activity of the nucleic acid (e.g., mRNA encodinga functional protein or enzyme) is prolonged over an extended period oftime. For example, the activity of the nucleic acids may be prolongedsuch that the compositions of the present invention are administered toa subject on a semi-weekly or bi-weekly basis, or more preferably on amonthly, bi-monthly, quarterly or an annual basis. The extended orprolonged activity of the compositions of the present invention, and inparticular of the mRNA comprised therein, is directly related to thequantity of functional protein or enzyme translated from such mRNA.Similarly, the activity of the compositions of the present invention maybe further extended or prolonged by modifications made to improve orenhance translation of the mRNA nucleic acids. For example, the Kozacconsensus sequence plays a role in the initiation of proteintranslation, and the inclusion of such a Kozac consensus sequence in themRNA nucleic acids of the present invention may further extend orprolong the activity of the mRNA nucleic acids. Furthermore, thequantity of functional protein or enzyme translated by the target cellis a function of the quantity of nucleic acid (e.g., mRNA) delivered tothe target cells and the stability of such nucleic acid. To the extentthat the stability of the nucleic acids of the present invention may beimproved or enhanced, the half-life, the activity of the translatedprotein or enzyme and the dosing frequency of the composition may befurther extended.

Accordingly, in a preferred embodiment, the nucleic acids providedherein comprise at least one modification which confers increased orenhanced stability to the nucleic acid, including, for example, improvedresistance to nuclease digestion in vivo. As used herein, the terms“modification” and “modified” as such terms relate to the nucleic acidsprovided herein, include at least one alteration which preferablyenhances stability and renders the nucleic acid more stable (e.g.,resistant to nuclease digestion) than the wild-type or naturallyoccurring version of the nucleic acid. As used herein, the terms“stable” and “stability” as such terms relate to the nucleic acids ofthe present invention, and particularly with respect to the mRNA, referto increased or enhanced resistance to degradation by, for examplenucleases (i.e., endonucleases or exonucleases) which are normallycapable of degrading such RNA. Increased stability can include, forexample, less sensitivity to hydrolysis or other destruction byendogenous enzymes (e.g., endonucleases or exonucleases) or conditionswithin the target cell or tissue, thereby increasing or enhancing theresidence of such nucleic acids in the target cell, tissue, subjectand/or cytoplasm. The stabilized nucleic acid molecules provided hereindemonstrate longer half-lives relative to their naturally occurring,unmodified counterparts (e.g., the wild-type version of the nucleicacid). Also contemplated by the terms “modification” and “modified” assuch terms related to the nucleic acids of the present invention arealterations which improve or enhance translation of mRNA nucleic acids,including for example, the inclusion of sequences which function in theinitiation of protein translation (e.g., the Kozac consensus sequence).(Kozak, M., Nucleic Acids Res 15 (20): 8125-48 (1987)).

In some embodiments, the nucleic acids of the present invention haveundergone a chemical or biological modification to render them morestable. Exemplary modifications to a nucleic acid include the depletionof a base (e.g., by deletion or by the substitution of one nucleotidefor another) or modification of a base, for example, the chemicalmodification of a base. The phrase “chemical modifications” as usedherein, includes modifications which introduce chemistries which differfrom those seen in naturally occurring nucleic acids, for example,covalent modifications such as the introduction of modified nucleotides,(e.g., nucleotide analogs, or the inclusion of pendant groups which arenot naturally found in such nucleic acid molecules).

In addition, suitable modifications include alterations in one or morenucleotides of a codon such that the codon encodes the same amino acidbut is more stable than the codon found in the wild-type version of thenucleic acid. For example, an inverse relationship between the stabilityof RNA and a higher number cytidines (C's) and/or uridines (U's)residues has been demonstrated, and RNA devoid of C and U residues havebeen found to be stable to most RNases (I leidenreich, et al. J BiolChem 269, 2131-8 (1994)). In some embodiments, the number of C and/or Uresidues in an mRNA sequence is reduced. In a another embodiment, thenumber of C and/or U residues is reduced by substitution of one codonencoding a particular amino acid for another codon encoding the same ora related amino acid. Contemplated modifications to the mRNA nucleicacids of the present invention also include the incorporation ofpseudouridines. The incorporation of pseudouridines into the mRNAnucleic acids of the present invention may enhance stability andtranslational capacity, as well as diminishing immunogenicity in vivo.(See, e.g., Kariko, K., et al., Molecular Therapy 16 (11): 1833-1840(2008)). Substitutions and modifications to the nucleic acids of thepresent invention may be performed by methods readily known to one orordinary skill in the art.

The constraints on reducing the number of C and U residues in a sequencewill likely be greater within the coding region of an mRNA, compared toan untranslated region, (i.e., it will likely not be possible toeliminate all of the C and U residues present in the message while stillretaining the ability of the message to encode the desired amino acidsequence). The degeneracy of the genetic code, however presents anopportunity to allow the number of C and/or U residues that are presentin the sequence to be reduced, while maintaining the same codingcapacity (i.e., depending on which amino acid is encoded by a codon,several different possibilities for modification of RNA sequences may bepossible). For example, the codons for Gly can be altered to GGA or GGGinstead of GGU or GGC.

The term modification also includes, for example, the incorporation ofnon-nucleotide linkages or modified nucleotides into the nucleic acidsequences of the present invention (e.g., modifications to one or boththe 3′ and 5′ ends of an mRNA molecule encoding a functional protein orenzyme). Such modifications include the addition of bases to a nucleicacid sequence (e.g., the inclusion of a poly A tail or a longer poly Atail), the alteration of the 3′ UTR or the 5′ UTR, complexing thenucleic acid with an agent (e.g., a protein or a complementary nucleicacid molecule), and inclusion of elements which change the structure ofa nucleic acid molecule (e.g., which form secondary structures).

The poly A tail is thought to stabilize natural messengers and syntheticsense RNA. Therefore, in one embodiment a long poly A tail can be addedto an mRNA molecule thus rendering the RNA more stable. Poly A tails canbe added using a variety of art-recognized techniques. For example, longpoly A tails can be added to synthetic or in vitro transcribed RNA usingpoly A polymerase (Yokoe, et al. Nature Biotechnology. 1996; 14:1252-1256). A transcription vector can also encode long poly A tails. Inaddition, poly A tails can be added by transcription directly from PCRproducts. Poly A may also be ligated to the 3′ end of a sense RNA withRNA ligase (see, e.g., Molecular Cloning A Laboratory Manual, 2nd Ed.,ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor LaboratoryPress: 1991 edition)). In one embodiment, the length of the poly A tailis at least about 90, 200, 300, 400, or at least about 500 nucleotides.In one embodiment, the length of the poly A tail is adjusted to controlthe stability of a modified sense mRNA molecule of the invention and,thus, the transcription of protein. For example, since the length of thepoly A tail can influence the half-life of a sense mRNA molecule, thelength of the poly A tail can be adjusted to modify the level ofresistance of the mRNA to nucleases and thereby control the time courseof protein expression in a cell. In one embodiment, the stabilizednucleic acid molecules are sufficiently resistant to in vivo degradation(e.g., by nucleases), such that they may be delivered to the target cellwithout a transfer vehicle.

In one embodiment, a nucleic acid encoding a protein can be modified bythe incorporation of 3′ and/or 5′ untranslated (UTR) sequences which Arenot naturally found in the wild-type nucleic acid. In one embodiment, 3′and/or 5′ flanking sequence which naturally flanks an mRNA and encodes asecond, unrelated protein can be incorporated into the nucleotidesequence of an mRNA molecule encoding a therapeutic or functionalprotein in order to modify it. For example, 3′ or 5′ sequences from mRNAmolecules which are stable (e.g., globin, actin, GAPDH, tubulin,histone, or citric acid cycle enzymes) can be incorporated into the 3′and/or 5′ region of a sense mRNA nucleic acid molecule to increase thestability of the sense mRNA molecule.

Also contemplated by the present invention are other modifications tothe nucleic acid sequences made to one or both of the 3′ and 5′ ends ofthe nucleic acid. For example, the present invention contemplatesmodifications to one or both of the 3′ and the 5′ ends of the nucleicacids (e.g., mRNA) to include at least a partial sequence of a CMVimmediate-early 1 (IE1) gene, or a fragment thereof (e.g., SEQ ID NO: 2or SEQ ID NO: 1) to improve the nuclease resistance and/or improve thehalf-life of the nucleic acid. In addition to increasing the stabilityof the mRNA nucleic acid sequence, it has been surprisingly discoveredthe inclusion of a partial sequence of a CMV immediate-early 1 (IE1)gene at the 5′ end enhances the translation of the mRNA and theexpression of the functional protein or enzyme. Also contemplated is theinclusion of a sequence encoding human growth hormone (hGH), or afragment thereof (e.g., SEQ ID NO: 3) to one or both of the 3′ and 5′ends of the nucleic acid (e.g., mRNA) to further stabilize the nucleicacid. Generally, preferred modifications improve the stability and/orpharmacokinetic properties (e.g., half-life) of the nucleic acidrelative to their unmodified counterparts, and include, for examplemodifications made to improve such nucleic acid's resistance to in vivonuclease digestion.

In some embodiments, the composition can comprise a stabilizing reagent.The compositions can include one or more formulation reagents that binddirectly or indirectly to, and stabilize the nucleic acid, therebyenhancing residence time in the cytoplasm of a target cell. Suchreagents preferably lead to an improved half-life of a nucleic acid inthe target cells. For example, the stability of an mRNA and efficiencyof translation may be increased by the incorporation of “stabilizingreagents” that form complexes with the nucleic acids (e.g., mRNA) thatnaturally occur within a cell (see e.g., U.S. Pat. No. 5,677,124).Incorporation of a stabilizing reagent can be accomplished for example,by combining the poly A and a protein with the mRNA to be stabilized invitro before loading or encapsulating the mRNA within a transfervehicle. Exemplary stabilizing reagents include one or more proteins,peptides, aptamers, translational accessory protein, mRNA bindingproteins, and/or translation initiation factors.

Stabilization of the compositions may also be improved by the use ofopsonization-inhibiting moieties, which are typically large hydrophilicpolymers that are chemically or physically bound to the transfer vehicle(e.g., by the intercalation of a lipid-soluble anchor into the membraneitself, or by binding directly to active groups of membrane lipids).These opsonization-inhibiting hydrophilic polymers form a protectivesurface layer which significantly decreases the uptake of the liposomesby the macrophage-monocyte system and reticulo-endothelial system (e.g.,as described in U.S. Pat. No. 4,920,016, the entire disclosure of whichis herein incorporated by reference). Transfer vehicles modified withopsonization-inhibition moieties thus remain in the circulation muchlonger than their unmodified counterparts.

When RNA is hybridized to a complementary nucleic acid molecule (e.g.,DNA or RNA) it may be protected from nucleases. (Krieg, et al. Melton.Methods in Enzymology. 1987; 155, 397-415). The stability of hybridizedmRNA is likely due to the inherent single strand specificity of mostRNases. In some embodiments, the stabilizing reagent selected to complexa nucleic acid is a eukaryotic protein, (e.g., a mammalian protein). Inyet another embodiment, the nucleic acid molecule (e.g., mRNA) for usein sense therapy can be modified by hybridization to a second nucleicacid molecule. If an entire mRNA molecule were hybridized to acomplementary nucleic acid molecule translation initiation may bereduced. Thus in some embodiments the 5′ untranslated region and the AUGstart region of the mRNA molecule may optionally be left unhybridized.Following translation initiation, the unwinding activity of the ribosomecomplex can function even on high affinity duplexes so that translationcan proceed. (Liebhaber. J. Mol. Biol. 1992; 226: 2-13; Monia, et al. JBiol Chem. 1993; 268: 14514-22.)

It will be understood that any of the above described methods forenhancing the stability of nucleic acids may be used either alone or incombination with one or more of 20 any of the other above-describedmethods and/or compositions.

In one embodiment, the compositions of the present invention facilitatethe delivery of nucleic acids to target cells. In some embodiments,facilitating delivery to target cells includes increasing the amount ofnucleic acid that comes in contact with the target cells. In someembodiments, facilitating delivery to target cells includes reducing theamount of nucleic acid that comes into contact with non-target cells. Insome embodiments, facilitating delivery to target cells includesallowing the transfection of at least some target cells with the nucleicacid. In some embodiments, the level of expression of the productencoded by the delivered nucleic acid is increased in target cells.

The nucleic acids of the present invention may be optionally combinedwith a reporter gene (e.g., upstream or downstream of the coding regionof the nucleic acid) which, for example, facilitates the determinationof nucleic acid delivery to the target cells or tissues. Suitablereporter genes may include, for example, Green Fluorescent Protein mRNA(GFP mRNA), Renilla Luciferase mRNA (Luciferase mRNA), FireflyLuciferase mRNA, or any combinations thereof. For example, GFP mRNA maybe fused with a nucleic acid encoding OTC mRNA to facilitateconfirmation of mRNA localization in the target cells, tissues ororgans.

As used herein, the terms “transfect” or “transfection” mean theintracellular introduction of a nucleic acid into a cell, or preferablyinto a target cell. The introduced nucleic acid may be stably ortransiently maintained in the target cell, The term “transfectionefficiency” refers to the relative amount of nucleic acid up-taken bythe target cell which is subject to transfection. In practice,transfection efficiency is estimated by the amount of a reporter nucleicacid product expressed by the target cells following transfection.Preferred are compositions with high transfection efficacies and inparticular those compositions that minimize adverse effects which aremediated by transfection of non-target cells and tissues.

As provided herein, the compositions can include a transfer vehicle. Asused herein, the term “transfer vehicle” includes any of the standardpharmaceutical carriers, diluents, excipients and the like which aregenerally intended for use in connection with the administration ofbiologically active agents, including nucleic acids. The compositionsand in particular the transfer vehicles described herein are capable ofdelivering nucleic acids of varying sizes to their target cells ortissues. In one embodiment of the present invention, the transfervehicles of the present invention are capable of delivering largenucleic acid sequences (e.g., nucleic acids of at least 1 kDa, 1.5 kDa,2 kDa, 2.5 kDa, SkDa, 10 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, ormore). The nucleic acids can be formulated with one or more acceptablereagents, which provide a vehicle for delivering such nucleic acids totarget cells. Appropriate reagents are generally selected with regardsto a number of factors, which include, among other things, thebiological or chemical properties of the nucleic acids (e.g., charge),the intended route of administration, the anticipated biologicalenvironment to which such nucleic acids will be exposed and the specificproperties of the intended target cells. In some embodiments, transfervehicles, such as liposomes, encapsulate the nucleic acids withoutcompromising biological activity. In some embodiments, the transfervehicle demonstrates preferential and/or substantial binding to a targetcell relative to non-target cells. In a preferred embodiment, thetransfer vehicle delivers its contents to the target cell such that thenucleic acids are delivered to the appropriate subcellular compartment,such as the cytoplasm.

In some embodiments, the transfer vehicle is a liposomal vesicle, orother means to facilitate the transfer of a nucleic acid to target cellsand tissues. Suitable transfer vehicles include, but are not limited to,liposomes, nanoliposomes, ceramide-containing nanoliposomes,proteoliposomes, nanoparticulates, calcium phosphor-silicatenanoparticulates, calcium phosphate nanoparticulates, silicon dioxidenanoparticulates, nanocrystalline particulates, semiconductornanoparticulates, poly(D-arginine), nanodendrimers, starch-baseddelivery systems, micelles, emulsions, niosomes, plasmids, viruses,calcium phosphate nucleotides, aptamers, peptides and other vectorialtags. Also contemplated is the use of bionanocapsules and other viralcapsid proteins assemblies as a suitable transfer vehicle. (Hum. GeneTher. 2008 September; 19(9):887-95). Also contemplated is the use ofpolymers as transfer vehicles, whether alone or in combination withother transfer vehicles. Suitable polymers may include, for example,polyacrylates, polyalkycyanoacrylates, polylactide,polylactide-polyglycolide copolymers, polycaprolactones, dextran,albumin, gelatin, alginate, collagen, chitosan, cyclodextrins andpolyethylenimine. In addition, several reagents are commerciallyavailable to enhance transfection efficacy. Suitable examples includeLIPOFECTIN (DOTMA:DOPE) (Invitrogen, Carlsbad, Calif.), LIPOFECTAMINE(DOSPA:DOPE) (Invitrogen), LIPOFECTAMINE2000. (Invitrogen), FUGENE,TRANSFECTAM (DOGS), and EFFECTENE. In one embodiment, the transfervehicle is selected based upon its ability to facilitate thetransfection of a nucleic acid to a target cell.

In one embodiment of the present invention, the transfer vehicle may beselected and/or prepared to optimize delivery of the nucleic acid to thetarget cell, tissue or organ. For example, if the target cell is ahepatocyte the properties of the transfer vehicle (e.g., size, chargeand/or pH) may be optimized to effectively deliver such transfer vehicleto the target cell or organ, reduce immune clearance and/or promoteretention in that target organ. Alternatively, if the target tissue isthe central nervous system (e.g., mRNA administered for the treatment ofneurodegenerative diseases may specifically target brain or spinaltissue) selection and preparation of the transfer vehicle must considerpenetration of, and retention within the blood brain barrier and/or theuse of alternate means of directly delivering such transfer vehicle tosuch target tissue. In one embodiment, the compositions of the presentinvention may be combined with agents that facilitate the transfer ofexogenous nucleic acids (e.g., agents which disrupt or improve thepermeability of the blood brain barrier and thereby enhance the transferof exogenous mRNA to the target cells).

In certain embodiments, the compositions of the present invention may beloaded with diagnostic radionuclide, fluorescent materials or othermaterials that are detectable in both in vitro and in vivo applications.For example, suitable diagnostic materials for use in the presentinvention may include Rhodamine-dioleoylphosphatidylethanolamine(Rh-PE), Green Fluorescent Protein mRNA (GFP mRNA), Renilla LuciferasemRNA and Firefly Luciferase mRNA.

In some embodiments, the compositions of the present invention compriseone or more additional molecules (e.g., proteins, peptides, aptamers oroliogonucleotides) which facilitate the transfer of the nucleic acids(e.g., mRNA, miRNA, snRNA and snoRNA) from the transfer vehicle into anintracellular compartment of the target cell. In one embodiment, theadditional molecule facilitates the delivery of the nucleic acids into,for example, the cytosol, the lysosome, the mitochondrion, the nucleus,the nucleolae or the proteasome of a target cell. Also included areagents that facilitate the transport of the translated protein ofinterest from the cytoplasm to its normal intercellular location (e.g.,in the mitochondrion) to treat deficiencies in that organelle. In someembodiments, the agent is selected from the group consisting of aprotein, a peptide, an aptamer, and an oligonucleotide.

In one embodiment, the compositions of the present invention facilitatea subject's endogenous production of one or more functional proteinsand/or enzymes, and in particular the production of proteins and/orenzymes which demonstrate less immunogenicity relative to theirrecombinantly-prepared counterparts. In a preferred embodiment of thepresent invention, the transfer vehicles comprise nucleic acids whichencode mRNA of a deficient protein or enzyme. Upon distribution of suchcompositions to the target tissues and the subsequent transfection ofsuch target cells, the exogenous mRNA may be translated in vivo toproduce a functional protein or enzyme encoded by the exogenouslyadministered mRNA (e.g., a protein or enzyme in which the subject isdeficient). Accordingly, the compositions of the present inventionexploit a subject's ability to translate exogenously- orrecombinantly-prepared mRNA to produce an endogenously-translatedprotein or enzyme, and thereby produce (and where applicable excrete) afunctional protein or enzyme. The expressed or translated proteins orenzymes may also be characterized by the in vivo inclusion of nativepost-translational modifications which may often be absent inrecombinantly-prepared proteins or enzymes, thereby further reducing theimmunogenicity of the translated protein or enzyme.

The administration of mRNA encoding a deficient protein or enzyme avoidsthe need to deliver the nucleic acids to specific organelles within atarget cell (e.g., mitochondria). Rather, upon transfection of a targetcell and delivery of the nucleic acids to the cytoplasm of the targetcell, the mRNA contents of a transfer vehicle may be translated and afunctional protein or enzyme expressed.

The present invention also contemplates the discriminatory targeting oftarget cells and tissues by both passive and active targeting means. Thephenomenon of passive targeting exploits the natural distributionspatterns of a transfer vehicle in vivo without relying upon the use ofadditional excipients or means to enhance recognition of the transfervehicle by target cells. For example, transfer vehicles which aresubject to phagocytosis by the cells of the reticulo-endothelial systemare likely to accumulate in the liver or spleen, and accordingly mayprovide means to passively direct the delivery of the compositions tosuch target cells.

Alternatively, the present invention contemplates active targeting,which involves the use of additional excipients, referred to herein as“targeting ligands” that may be bound (either covalently ornon-covalently) to the transfer vehicle to encourage localization ofsuch transfer vehicle at certain target cells or target tissues. Forexample, targeting may be mediated by the inclusion of one or moreendogenous targeting ligands (e.g., apolipoprotein E) in or on thetransfer vehicle to encourage distribution to the target cells ortissues. Recognition of the targeting ligand by the target tissuesactively facilitates tissue distribution and cellular uptake of thetransfer vehicle and/or its contents in the target cells and tissues(e.g., the inclusion of an apolipoprotein-E targeting ligand in or onthe transfer vehicle encourages recognition and binding of the transfervehicle to endogenous low density lipoprotein receptors expressed byhepatocytes). As provided herein, the composition can comprise a ligandcapable of enhancing affinity of the composition to the target cell.These methods are well known in the art. In other some embodiments, thecompositions of the present invention demonstrate improved transfectionefficacies, and/or demonstrate enhanced selectivity towards target cellsor tissues of interest. Contemplated therefore are compositions whichcomprise one or more ligands (e.g., peptides, aptamers,oligonucleotides, a vitamin or other molecules) that are capable ofenhancing the affinity of the compositions and their nucleic acidcontents for the target cells or tissues. Suitable ligands mayoptionally be bound or linked to the surface of the transfer vehicle. Insome embodiments, the targeting ligand may span the surface of atransfer vehicle or be encapsulated within the transfer vehicle.Suitable ligands and are selected based upon their physical, chemical orbiological properties (e.g., selective affinity and/or recognition oftarget cell surface markers or features.) Cell specific target sites andtheir corresponding targeting ligand can vary widely. Suitable targetingligands are selected such that the unique characteristics of a targetcell are exploited, thus allowing the composition to discriminatebetween target and non-target cells. For example, compositions of thepresent invention may bear surface markers (e.g., apolipoprotein-B orapolipoprotein-E) that selectively enhance recognition of, or affinityto hepatocytes (e.g., by receptor-mediated recognition of and binding tosuch surface markers). Additionally, the use of galactose as a targetingligand would be expected to direct the compositions of the presentinvention to parenchymal hepatocytes, or alternatively the use ofmannose containing sugar residues as a targeting ligand would beexpected to direct the compositions of the present invention to liverendothelial cells (e.g., mannose containing sugar residues that may bindpreferentially to the asialoglycoprotein receptor present inhepatocytes). (See Hillery A M, el al. “Drug Delivery and Targeting: ForPharmacists and Pharmaceutical Scientists” (2002) Taylor & Francis,Inc.) The presentation of such targeting ligands therefore facilitaterecognition and uptake of the compositions of the present invention intarget cells and tissues. Examples of suitable targeting ligands includeone or more peptides, proteins, aptamers, vitamins and oligonucleotides.

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,rodents, and the like, to which the compositions and methods of thepresent invention are administered. Typically, the terms “subject” and“patient” are used interchangeably herein in reference to a humansubject.

As used herein, the term “target cell” refers to a cell or tissue towhich a composition of the invention is to be directed or targeted. Insome embodiments, the target cells are deficient in a protein or enzymeof interest. For example, where it is desired to deliver a nucleic acidto a hepatocyte, the hepatocyte represents the target cell.

In some embodiments, the nucleic acids and compositions of the presentinvention transfect the target cells on a discriminatory basis (i.e., donot transfect non-target cells). The compositions and methods of thepresent invention may be prepared to preferentially target a variety oftarget cells, which include, but are not limited to, hepatocytes,epithelial cells, hematopoietic cells, epithelial cells, endothelialcells, lung cells, bone cells, stem cells, mesenchymal cells, neuralcells (e.g., meninges, astrocytes, motor neurons, cells of the dorsalroot ganglia and anterior horn motor neurons), photoreceptor cells(e.g., rods and cones), retinal pigmented epithelial cells, secretorycells, cardiac cells, adipocytes, vascular smooth muscle cells,cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells,synovial lining cells, ovarian cells, testicular cells, fibroblasts, Bcells, T cells, reticulocytes, leukocytes, granulocytes and tumor cells.

Following transfection of one or more target cells by the compositionsand nucleic acids of the present invention, expression of the proteinencoded by such nucleic acid may be preferably stimulated and thecapability of such target cells to express the protein of interest isenhanced. For example, transfection of a target cell with an OTC mRNAwill allow expression of the OTC protein product following translationof the nucleic acid. The nucleic acids of the compositions and/ormethods provided herein preferably encode a product (e.g., a protein,enzyme, polypeptide, peptide, functional RNA, and/or antisensemolecule), and preferably encode a product whose in vivo production isdesired.

The urea cycle metabolic disorders represent examples of protein andenzyme deficiencies which may be treated using the methods andcompositions provided herein. Such urea cycle metabolic disordersinclude OTC deficiency, arginosuccinate synthetase deficiency (ASD) andargininosuccinate lyase deficiency (ALD). Therefore, in someembodiments, the nucleic acid of the methods and compositions providedherein encode an enzyme involved in the urea cycle, including, forexample, omithine transcarbamylase (OTC), carbamyl phosphate synthetase(CPS), argininosuccinate synthetase 1 (ASS1) argininosuccinate lyase(ASL), and arginase (ARG).

Five metabolic disorders which result from defects in the biosynthesisof the enzymes involved in the urea cycle have been described, andinclude ornithine transcarbamylase (OTC) deficiency, carbamyl phosphatesynthetase (CPS) deficiency, argininosuccinate synthetase 1 (ASS1)deficiency (citrullinemia), argininosuccinate lyase (ASL) deficiency andarginase deficiency (ARG). Of these five metabolic disorders, OTCdeficiency represents the most common, occurring in an estimated one outof every 80,000 births.

OTC is a homotrimeric mitochondrial enzyme which is expressed almostexclusively in the liver and which encodes a precursor OTC protein thatis cleaved in two steps upon incorporation into the mitchondrial matrix.(Horwich A L., et al. Cell 1986; 44: 451-459). OTC deficiency is agenetic disorder which results in a mutated and biologically inactiveform of the enzyme ornithine transcarbamylase. OTC deficiency oftenbecomes evident in the first few days of life, typically after proteiningestion. In the classic severe form of OTC deficiency, within thefirst days of life patients present with lethargy, convulsions, coma andsevere hyperammonemia, which quickly leads to a deteriorating and fataloutcome absent appropriate medical intervention. (Monish S., et al.,Genetics for Pediatricians; Remedica, Cold Spring Harbor Laboratory(2005)). If improperly treated or if left untreated, complications fromOTC deficiency may include developmental delay and mental retardation.OTC deficient subjects may also present with progressive liver damage,skin lesions, and brittle hair. In some affected individuals, signs andsymptoms of OTC deficiency may be less severe, and may not appear untillater in life.

The OTC gene, which is located on the short arm of the X chromosomewithin band Xp21.1, spans more than 85 kb and is comprised of 10 exonsencoding a protein of 1062 amino acids. (Lindgren V., et al. Science1984; 226: 698-7700; Horwich, A L., et al. Science 224: 1068-1074, 1984;Horwich, A L. et al., Cell 44: 451-459, 1986; Hata, A., et al., J.Biochem. 100: 717-725, 1986, which are incorporated herein byreference). The OTC enzyme catalyzes the conversion or onnthine andcarbamoyl phosphate to citrulline. Since OTC is on the X chromosome,females are primarily carriers while males with nonconservativemutations rarely survive past 72 hours of birth.

In healthy subjects, 07'C is expressed almost exclusively inhepatocellular mitochondria. Although not expressed in the brain ofhealthy subjects, OTC deficiency can lead to neurological disorders. Forexample, one of the usual symptoms of OTC deficiency, which isheterogeneous in its presentation, is hyperammonaemic coma (Gordon, N.,Eur J Paediatr Neurol 2003; 7:115-121.).

OTC deficiency is very heterogeneous, with over 200 unique mutationsreported and large deletions that account for approximately 10-15% ofall mutations, while the remainder generally comprises missense pointmutations with smaller numbers of nonsense, splice-site and smalldeletion mutations. (Monish A., et al.) The phenotype of OTC deficiencyis extremely heterogeneous, which can range from acute neonatalhyperammonernie coma to asymptomatic hemizygous adults. (Gordon N. Eur JPaediatr Neurol 2003; 7: 115-121). Those mutations that result in severeand life threatening neonatal disease are clustered in importantstructural and flinctional domains in the interior of the protein atsites of enzyme activity or at the interchain surface, while mutationsassociated with late-onset disease are located on the protein surface(Monish A., et al.) Patients with milder or partial forms of OTCdeficiency may have onset of disease later in life, which may present asrecurrent vomiting, neurobehavioral changes or seizures associated withhyperammonemia.

The compositions and methods of the present invention are broadlyapplicable to the delivery of nucleic acids, and in particular mRNA, totreat a number of disorders. In particular, the compositions and methodsof the present invention are suitable for the treatment of diseases ordisorders relating to the deficiency of proteins and/or enzymes. In oneembodiment, the nucleic acids of the present invention encode functionalproteins or enzymes that are excreted or secreted by the target cellinto the surrounding extracellular fluid (e.g., mRNA encoding hormonesand neurotransmitters). Alternatively, in another embodiment, thenucleic acids of the present invention encode functional proteins orenzymes that remain in the cytosol of the target cell (e.g., mRNAencoding urea cycle metabolic disorders).

Other disorders for which the present invention can be useful as atherapeutic intervention include enzyme and protein deficiencies, suchas lysosomal storage diseases. Specific disorders for which the presentinvention can be useful as a therapeutic intervention include disorderssuch as SMN I-related spinal muscular atrophy (SMA); amyotrophic lateralsclerosis (ALS); GALT-related galactosemia; Cystic Fibrosis (CF);SLC3A1-related disorders including cystinuria; COL4A5-related disordersincluding Alport syndrome; galactocerebrosidase deficiencies; X-linkedadrenoleukodystrophy and adrenomyeloneuropathy; Friedreich's ataxia;Pelizaeus-Merzbacher disease; TSC1 and TSC2-related tuberous sclerosis;Sanfilippo B syndrome (MPS IIIB); CTNS-related cystinosis; theFMR1-related disorders which include Fragile X syndrome, FragileX-Associated Tremor/Ataxia Syndrome and Fragile X Premature OvarianFailure Syndrome; Prader-Willi syndrome; hereditary hemorrhagictelangiectasia (AT); Niemann-Pick disease Type C1; the neuronal ceroidlipofuscinoses-related diseases including Juvenile Neuronal CeroidLipofuscinosis (JNCL), Juvenile Batten disease, Santavuori-Haltiadisease, Jansky-Bielschowsky disease, and PTT-1 and TPP1 deficiencies;EIF2B1, EIF2B2, EIF2B3, EIF2B4 and E1F2B5-related childhood ataxia withcentral nervous system hypomyelination/vanishin white matter; CACNA IAand CACNB4-related Episodic Ataxia Type 2; the MECP2-related disordersincluding Classic Rett Syndrome, MECP2-related Severe NeonatalEncephalopathy and PPM-X Syndrome; CDKL5-related Atypical Rett Syndrome;Kennedy's disease (SBMA); Notch-3 related cerebral autosomal dominantarteriopathy with subcortical infarcts and leukoencephalopathy(CADASIL); SCN1A and SCNIB-related seizure disorders; the PolymeraseG-related disorders which include Alpers-Huttenlocher syndrome,POLG-related sensory ataxic neuropathy, dysarthria, andophthalmoparesis, and autosomal dominant and recessive progressiveexternal ophthalmoplegia with mitochondrial DNA deletions; X-Linkedadrenal hypoplasia; X-linked agammaglobulinemia; Fabry disease; andWilson's disease.

In one embodiment, the nucleic acids, and in particular mRNA, of thepresent invention may encode functional proteins or enzythes. Forexample, the compositions of the present invention may include mRNAencoding erythropoietin (EPO), a I-antitrypsin, carboxypeptidase N,alpha galactosidase (GLA), ornithine carbamoyltransferase (OTC), orhuman growth hormone (hOtt).

Alternatively the nucleic acids may encode full length antibodies orsmaller antibodies (e.g., both heavy and light chains) to conferimmunity to a subject. While one embodiment of the present inventionrelates to methods and compositions useful for confen-ing immunity to asubject (e.g., via the translation of mRNA nucleic acids encodingfunctional antibodies), the inventions disclosed herein and contemplatedhereby are broadly applicable. In an alternative embodiment thecompositions of the present invention encode antibodies that may be usedto transiently or chronically effect a functional response in subjects.For example, the mRNA nucleic acids of the present invention may encodea functional monoclonal or polyclonal, antibody, which upon translation(and as applicable, systemic excretion from the target cells) may beuseful for targeting and/or inactivating a biological target (e.g., astimulatory cytokine such as tumor necrosis factor). Similarly, the mRNAnucleic acids of the present invention may encode, for example,functional anti-nephritic factor antibodies useful for the treatment ofmembranoproliferative glomerulonephritis type II or acute hemolyticuremic syndrome, or alternatively may encode anti-vascular endothelialgrowth factor (VEGF) antibodies useful for the treatment ofVEGF-mediated diseases, such as cancer.

The compositions of the present invention can be administered to asubject. In some embodiments, the composition is formulated incombination with one or more additional nucleic acids, carriers,targeting ligands or stabilizing reagents, or in pharmacologicalcompositions where it is mixed with suitable excipients. For example, inone embodiment, the compositions of the present invention may beprepared to deliver nucleic acids (e.g., mRNA) encoding two or moredistinct proteins or enzymes. Alternatively, the compositions of thepresent invention may be prepared to deliver a single nucleic acid andtwo or more populations or such compositions may be combined in a singledosage form or co-administered to a subject. Techniques for formulationand administration of drugs may be found in “Remington's PharmaceuticalSciences,” Mack Publishing Co., Easton, Pa., latest edition.

A wide range of molecules that can exert pharmaceutical or therapeuticeffects can be delivered into target cells using compositions andmethods of the present invention. The molecules can be organic orinorganic. Organic molecules can be peptides, proteins, carbohydrates,lipids, sterols, nucleic acids (including peptide nucleic acids), or anycombination thereof. A formulation for delivery into target cells cancomprise more than one type of molecule, for example, two differentnucleotide sequences, or a protein, an enzyme or a steroid.

The compositions of the present invention may be administered and dosedin accordance with current medical practice, taking into account theclinical condition of the subject, the site and method ofadministration, the scheduling of administration, the subject's age,sex, body weight and other factors relevant to clinicians of ordinaryskill in the art. The “effective amount” for the purposes herein may bedetermined by such relevant considerations as are known to those ofordinary skill in experimental clinical research, pharmacological,clinical and medical arts. In some embodiments, the amount administeredis effective to achieve at least some stabilization, improvement orelimination of symptoms and other indicators as are selected asappropriate measures of disease progress, regression or improvement bythose of skill in the art. For example, a suitable amount and dosingregimen is one that causes at least transient expression of the nucleicacid in the target cell.

Suitable routes of administration include, for example, oral, rectal,vaginal, transmucosal, or intestinal administration; parenteraldelivery, including intramuscular, subcutaneous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intravenous, intraperitoneal, intranasal, or intraocular injections.

Alternately, the compositions of the present invention may beadministered in a local rather than systemic manner, for example, viainjection of the pharmaceutical composition directly into a targetedtissue, preferably in a depot or sustained release formulation. Localdelivery can be affected in various ways, depending on the tissue to betargeted. For example, aerosols containing compositions of the presentinvention can be inhaled (for nasal, tracheal, or bronchial delivery);compositions of the present invention can be injected into the site ofinjury, disease manifestation, or pain, for example; compositions can beprovided in lozenges for oral, tracheal, or esophageal application; canbe supplied in liquid, tablet or capsule form for administration to thestomach or intestines, can be supplied in suppository form for rectal orvaginal application; or can even be delivered to the eye by use ofcreams, drops, or even injection. Formulations containing compositionsof the present invention complexed with therapeutic molecules or ligandscan even be surgically administered, for example in association with apolymer or other structure or substance that can allow the compositionsto diffuse from the site of implantation to surrounding cells.Alternatively, they can be applied surgically without the use ofpolymers or supports.

In one embodiment, the compositions of the present invention areformulated such that they are suitable for extended-release of thenucleic acids contained therein. Such extended-release compositions maybe conveniently administered to a subject at extended dosing intervals.For example, in one embodiment, the compositions of the presentinvention are administered to a subject twice day, daily or every otherday. In a preferred embodiment, the compositions of the presentinvention are administered to a subject twice a week, once a week, everyten days, every two weeks, every three weeks, or more preferably everyfour weeks, once a month, every six weeks, every eight weeks, everyother month, every three months, every four months, every six months,every eight months, every nine months or annually. Also contemplated arecompositions and liposomal vehicles which are formulated for depotadministration (e.g., intramuscularly, subcutaneously, intravitreally)to either deliver or release a nucleic acids (e.g., mRNA) over extendedperiods of time. Preferably, the extended-release means employed arecombined with modifications made to the nucleic acid to enhancestability.

While certain compounds, compositions and methods of the presentinvention have been described with specificity in accordance withcertain embodiments, the following examples serve only to illustrate thecompounds of the invention and are not intended to limit the same. Eachof the publications, reference materials, accession numbers and the likereferenced herein to describe the background of the invention and toprovide additional detail regarding its practice are hereby incorporatedby reference in their entirety.

The articles “a” and “an” as used herein in the specification and in theclaims, unless clearly indicated to the contrary, should be understoodto include the plural referents. Claims or descriptions that include“or” between one or more members of a group are considered satisfied ifone, more than one, or all of the group members are present in, employedin, or otherwise relevant to a given product or process unless indicatedto the contrary or otherwise evident from the context. The inventionincludes embodiments in which exactly one member of the group is presentin, employed in, or otherwise relevant to a given product or process.The invention also includes embodiments in which more than one, or theentire group members are present in, employed in, or otherwise relevantto a given product or process. Furthermore, it is to be understood thatthe invention encompasses all variations, combinations, and permutationsin which one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim dependent on the same base claim (or, as relevant, any otherclaim) unless otherwise indicated or unless it would be evident to oneof ordinary skill in the art that a contradiction or inconsistency'would arise. Where elements are presented as lists, (e.g., in Markushgroup or similar format) it is to be understood that each subgroup ofthe elements is also disclosed, and any element(s) can be removed fromthe group. It should be understood that, in general, where theinvention, or aspects of the invention, is/are referred to as comprisingparticular elements, features, etc., certain embodiments of theinvention or aspects of the invention consist, or consist essentiallyof, such elements, features, etc. For purposes of simplicity thoseembodiments have not in every case been specifically set forth in somany words herein. It should also be understood that any embodiment oraspect of the invention can be explicitly excluded from the claims,regardless of whether the specific exclusion is recited in thespecification. The publications and other reference materials referencedherein to describe the background of the invention and to provideadditional detail regarding its practice are hereby incorporated byreference.

1-20. (canceled)
 21. A method of treating a urea cycle disordercomprising administering to a subject suffering from or susceptible to aurea cycle disorder, a composition comprising an mRNA encoding anenzyme, encapsulated within a liposome; wherein the enzyme is selectedfrom ornithine transcarbamylase (OTC), carbamyl phosphate synthetase(CPS), argininosuccinate synthetase 1 (ASS1) argininosuccinate lyase(ASL), or arginase (ARG).
 22. The method of claim 21, wherein the enzymeis ASS1.
 23. The method of claim 21, wherein the mRNA is administeredsystemically.
 24. The method of claim 21, wherein the composition isadministered intravenously.
 25. The method of claim 21, wherein thecomposition is administered bi-weekly.
 26. The method of claim 21,wherein the composition is administered monthly.
 27. The method of claim21, wherein the administering of the composition results in increasedexpression of the enzyme encoded by the mRNA in the liver.
 28. Themethod of claim 21, wherein the mRNA comprises a modification to enhancestability.
 29. The method of claim 28, wherein the modification isselected from a group consisting of a modified nucleotide, an alterationto the 5′ or 3′ untranslated region (UTR), a cap structure and a poly Atail.
 30. The method of claim 29, wherein the modification comprises apseudouridine.
 31. The method of claim 29, wherein the modificationcomprises a partial sequence of a CMV immediate-early 1 (IE1) gene. 32.The method of claim 31, wherein said partial sequence of the CMVimmediate-early (IE1) gene comprises SEQ ID NO: 1, wherein SEQ ID NO: 1enhances the expression of the protein of interest.